A breakdown in self-tolerance can result in the immune system raising an arsenal against the body's own defenses leading to diseases caused by inappropriate T lymphocyte (T-cell) responses. These include autoimmune diseases (multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, lupus erythromatosis, etc.) and chronic inflammatory diseases. Similarly, graft rejection following transplant surgery is a significant clinical issue and arises due to recognition of foreign antigens from the graft by the host immune system. As T-cells are the key regulators of these immune system assaults, an inhibitor of T-cell function should have broad application as therapeutic agents in these diseases. Currently, the leading medicinal agent for the treatment of graft rejection is cyclosporin A (CsA), approved by the Food and Drug Administration in 1983. CsA inhibits the catalytic function of calcineurin, a phosphatase that plays a key role in signal transduction from the T-cell receptor (TcR) to the nucleus. Calcineurin is ubiquitously expressed and is involved in many other transduction pathways. As a result, CsA has a very narrow therapeutic index and suffers from its propensity to cause kidney failure, liver damage and ulcers. Safer drugs that are able to modulate the immune response with fewer side effects are needed.
Lck (lymphocyte cell kinase), a Src-family protein tyrosine kinase expressed primarily in T-cells, plays an essential role in the immune response. Crucially, Lck is upstream of calcineurin in the TcR signaling cascade. Productive antigen-induced T-cell activation is characterized by the appearance of a Lck-driven, hyperphosphorylated TcR ζ chain and by phosphorylation-dependent catalytic activation of the Syk-family kinase ZAP-70 by Lck after docking of ZAP-70 tandem SH2 domains to the phosphorylated amino acids (ITAM motif) in the ζ chain. Activated ZAP-70 phosphorylates several substrates that serve as adapter proteins for binding of downstream signaling molecules. This signaling cascade culminates in transcriptional activation of genes involved in cytokine release (particularly IL-2), and ultimately in T-cell clonal expansion in response to an autocrine growth pathway as a prelude to raising an immune response.
Lck is one of eight known members of the human Src-family of protein tyrosine kinases, the others being Src, Fyn, Lyn, Hck, Blk, Yes and Fgr. As a consequence of alternate mRNA splicing, Fyn exists as two distinct gene products, Fyn(T) and Fyn(B) that differ in their ATP binding sites. All Src-family kinases have a similar structure, comprised of an N-terminal Src-homology (“SH”) 4 (“SH4”) domain, a “unique” domain, an SH3 domain, an SH2 domain, a catalytic domain (also known as the SH1 domain or the kinase domain) and a short C-terminal tail. Activity is regulated by tyrosine phosphorylation at two sites. Phosphorylation of a tyrosine (Tyr-505, Src numbering) in the C-terminal tail leads to down-regulation by promoting an intramolecular interaction between the tail and the SH2 domain. In vivo, the protein phosphatase CD-45 is thought to dephosphorylate this terminal tyrosine to allow autophosphorylation of a tyrosine (Tyr-394) in the activation loop segment of the kinase domain to generate catalytically-competent Lck.
The eight known mammalian members of the Src-family break down into two sub-families. Lck is most similar to Hck, Lyn and Blk (identities greater than 65% between any two members). The other sub-family consists of Src, Yes, Fyn and Fgr (identities greater than 70% between any two members). These kinases have higher similarity when the catalytic domains alone are compared (in some cases greater than 90%). Residues that are important for Src-family kinase activity and/or substrate specificity have been identified by X-ray crystal structures and by structural modeling studies, and are highly conserved among family members. This high level of similarity presents a challenge for designing even partially specific inhibitors.
Genetic data clearly validate Lck as a target. Severe Combined Immune Deficiency (SCID)-like phenotypes have been observed in mice rendered Lck-deficient by homologous recombination. Individuals with mutations in the gene encoding ZAP-70 have been identified and present with an absence of peripheral CD8+ T-cells and normal levels of peripheral CD4+ T-cells that are unable to signal through the TcR. A single instance of a human disease-associated defect in Lck expression has been reported. The infant described exhibits the clinical features of SCID, has selective CD4+ lymphopenia and lacks expression of the CD28 co-stimulatory molecule on CD8+ T-cells. Lck protein in the patient is expressed at <10% of the level observed in control T-cells. T-cells from this patient had defective proliferative responses to mitogens and IL-2, while some TcR proximal signaling events (e.g. mobilization of intracellular calcium) did not seem to be impaired.
Selective and non-selective kinase inhibitors have been shown to block T-cell receptor-dependent effects in cellular assays, thus validating inhibitors as modulators of T-cell function. Given the genetic and pharmacologic data for the role of Lck in T-cell activation, Lck appears to be a target suited to therapeutic intervention in indications where the disease process is T-cell dependent. Selective inhibition of Lck function therefore represents an attractive target for therapeutic intervention in the treatment of autoimmune and inflammatory diseases and also in organ transplantation. Given the very restricted cellular expression pattern for the target enzyme, the mechanism-based toxicity of selective Lck inhibitors should result in fewer side effects than cyclosporine A or corticosteroids.
Several crystal structures have been reported of Src-family protein kinases. Among these are:                1. a structure of the catalytic domain of human Lck in the activated state, that is, phosphorylated on Tyr-394 in the activation loop (Yamaguchi & Hendrickson, 1996);        2. a structure of human Src (SH3, SH2, catalytic domain, and C-terminal tail) in the autoinhibited state, that is not phosphorylated on Tyr-416 in the activation loop, but instead phosphorylated on Tyr-527 in the C-terminal tail (numbering of amino acid residues corresponds to the gene for chicken c-Src) (Xu et al., 1997);        3. a structure of human Hck (SH3, SH2, catalytic domain, and C-terminal tail) in the autoinhibited state, that is not phosphorylated on Tyr-416 in the activation loop, but instead phosphorylated on Tyr-527 in the C-terminal tail (numbering of amino acid residues corresponds to the gene for chicken c-Src) (Sicheri et al., 1997);        4. a structure of an autoinhibited human Hck/ligand complex (ligand PP1) (Schindler et al., 1999);        5. five structures of human Src (SH3, SH2, catalytic domain, and C-terminal tail) in the autoinhibited state, that is not phosphorylated on Tyr-416 in the activation loop, but instead phosphorylated on Tyr-527 in the C-terminal tail (numbering of amino acid residues corresponds to the gene for chicken c-Src), of which one structure is a Src/ligand complex (ligand AMP-PNP) (Xu et al., 1999);        6. a structure of chicken Src (SH3, SH2, catalytic domain, and C-terminal tail) in the autoinhibited state, that is not phosphorylated on Tyr-416 in the activation loop, but instead phosphorylated on Tyr-527 in the C-terminal tail (Williams et al., 1997);        7. three structures of the catalytic domain of activated human Lck/ligand complexes (ligands AMP-PNP, staurosporine, and PP2) (Zhu et al., 1999).        
Crystal structures have been determined also for the kinase domains of a wide variety of protein tyrosine and serine/threonine kinases outside of the Src-family, for example Abl (Schindler et al., 2000), Tie2 (Shewchuk et al., 2000), insulin receptor (Hubbard et al., 1994), FGF receptor (Mohammadi et al., 1996), VEGF receptor (McTigue et al., 1999), cAMP-dependent protein kinase (Knighton et al., 1991), cyclin-dependent kinase 2 (Cdk2) (Schulze-Gahmen et al., 1996), PAK1 (Lei et al., 2000), GSK-3β (Dajani et al., 2001), among others.
Crystal structures have also been determined for the kinase domains of certain protein kinases complexed to other proteins, for example Cdk2 complexed to cyclin A (Chan et al., 2001), and Cdk2 complexed to cyclin A and p27Kip1 (Russo et al., 1996), among others.
In addition, crystal structures have been determined for certain non-catalytic domains of some protein kinases, for example a regulatory subunit of cAMP-dependent protein kinase (Su et al., 1995), an SH2 domain of a Src-family protein tyrosine kinase (Waksman et al., 1992), and an SH3 domain of a Src-family protein tyrosine kinase (Noble et al., 1993), among others.
Three-dimensional structures for certain non-catalytic domains of some protein kinases have also been determined using other techniques, such as nuclear magnetic resonance (NMR). Examples include an SH2 domain of Syk (Narula et al., 1995) and the SH2 and SH3 domains of Abl (Gosser et al., 1995), among others.
Previously determined crystal structures of Src-family protein tyrosine kinases, especially those of Lck, Hck, and Src, all suffer defects that limit their usefulness in guiding the design of improved inhibitors. These limitations include, among others:                1. structures determined without ligands bound to the protein tyrosine kinase, for example the structure of the catalytic domain of human Lck in the activated state referred to above (Yamaguchi & Hendrickson, 1996);        2. structures determined of kinase/ligand complexes wherein the ligands bind weakly to the kinase, for example the structure of an activated human Lck catalytic domain/ligand complex (ligand AMP-PNP) referred to above (Zhu et al., 1999);        3. structures determined of kinase/ligand complexes wherein the ligands exhibit non-specific binding to a variety of kinases, for example the structure of an autoinhibited human Hck/ligand complex (ligand PP1) referred to above (Schindler et al., 1999);        4. structures determined of kinase/ligand complexes wherein potential ligand binding sites in the kinase catalytic domain are not accessed by the ligands, for example the structure of an autoinhibited human Hck/ligand complex (ligand PP1) referred to above (Schindler et al., 1999), as well as the three structures of activated human Lck catalytic domain/ligand complexes (ligands AMP-PNP, staurosporine, and PP2) referred to above (Zhu et al., 1999). These structures do not teach how a ligand should be designed in order to best interact with potential binding sites.        
A further limitation of the prior art has been that the structures of Src-family protein tyrosine kinases referred to above, especially those of Lck, Hck, and Src, all were determined using catalytically-active enzymes. The intrinsic catalytic activity of these kinases limits which phosphorylation states of the kinase are experimentally-accessible. It is well-known that the regulation of Src-family protein tyrosine kinases is regulated in part by differential phosphorylation (Superti-Furga, 1995).
Yet another limitation of the prior art has been that all previous crystal structures of Lck have been determined using an activated Lck catalytic domain that is phosphorylated at Tyr-394 (Yamaguchi & Hendrickson, 1996; Zhu et al., 1999). Other prior work on another Src-family protein tyrosine kinase, Hck, has demonstrated, however, that the phosphorylation state of the corresponding residue in Hck (Tyr-416; numbering of amino acid residues corresponds to the gene for chicken c-Src) likely alters the ability of Hck to bind ligands (Schindler et al., 1999). While this latter Hck crystal structure does not teach how a ligand should be designed in order to best interact with potential binding sites on Hck, let alone Lck, it is clear that experimental access to crystal structures of Src-family protein tyrosine kinases in several different phosphorylation states is desirable, but not yet achieved. Furthermore, it is not clear which phosphorylation state of a Src-family protein tyrosine kinase such as Lck is the therapeutically-relevant target for inhibition, or indeed whether several different phosphorylation states are all therapeutically-relevant targets, but under different conditions (such as disease state, tissue, etc.).
A final limitation of the prior art has been that all previous crystal structures of Lck, Hck, and Src determined as a kinase/ligand complex have been determined with inhibitors that do not access or contact amino acid residues that are unique to that particular kinase within the Src family. Examples include the structure of an autoinhibited human Hck/ligand complex (ligand PP1) referred to above (Schindler et al., 1999); the three structures of activated human Lck catalytic domain/ligand complexes (ligands AMP-PNP, staurosporine, and PP2) referred to above (Zhu et al., 1999); and the structure of an autoinhibited Src/ligand complex (ligand AMP-PNP) referred to above (Xu et al., 1999). Thus, these structures do not teach how a ligand should be designed in order to best interact with unique binding sites that could provide binding selectivity within the Src family.
Due to its role in T cell-mediated immune responses, Lck is a potential target for therapies aimed at controlling autoimmune and inflammatory diseases, cancer and also in treating organ transplant rejection. The development of biochemical assays for Lck has enabled drug discovery to proceed along the pathways of identifying lead Lck inhibitors by high-throughput screening of compound libraries and by testing compounds that mimic substrate structure. As discussed above, however, rational, structure-based design has not been possible up to this point because of the lack of accurate three-dimensional structural data for Lck complexed to appropriate ligands.